The asialoglycoprotein (ASGP) receptor is a transmembrane glycoprotein specific to hepatocytes which has been used as a model system to explore receptor biosynthesis, trafficking and distribution within endosomal compartments. In addition, the ASGP receptor in vivo has been used as a target for liver-specific gene transformation suggesting a potential application to the therapy of inherited metabolic diseases. This proposal investigates the mechanisms regulating the cell surface expression and bioactivity of this model cell surface receptor. The specific aims of this project focus on receptor phosphorylation and its relationship to the regulation of receptor trafficking and down-regulation. This hypothesis was suggested by data on the ASGP and other receptors which are phosphorylated during internalization and down-regulation (beta adrenergic receptor, insulin, epidermal growth factor receptor). Prior studies from this laboratory identified an intracellular population of phosphorylated ASGP receptors which does not recycle to plasma membrane. The physiologic role and regulation of this intracellular population of receptors will be investigated in the Hep G2 cell line. The experimental approaches to this question will include whole cell studies in Hep G2 cells, membrane preparations, and mammalian cell lives transfected with wild-type ASGP receptor cDNA and that mutated at the identified phosphorylation sites. Several novel techniques in whole cells (selective immunoprecipitation of surface receptors, density-shift analysis of isolated endosomal vesicles, and biotinylation of cell surface receptors) will address the subcellular location of the initial phosphorylation event, the routing of phosphorylated molecules in intracellular compartments. Tryptic phosphopeptide and phosphoamino acid analysis will localize the site of phosphorylation on the receptor polypeptide. Site-directed mutagenesis will then be employed to specifically alter the amino acid at this site, creating phosphorylation- negative ASGP receptor mutants which will be transfected into suitable surrogate cells. These cell lines expressing mutated and non mutated receptor will be analyzed trafficking pathways. The combination of these independent methodological approaches in this model system should yield information on the relevance of phosphorylation to receptor regulation in hepatocytes.